This work was supported by National Institutes of Health grants F32 NS078958 (DTB) and R01 AG033620 (BG).

1. Assemble Flight Tester
<ol>
	<li>Secure flight cylinder to Ring Stand 1 using chain clamps. (Leave approximately 3 cm underneath the cylinder for weigh dish.) 
	(Note: The flight cylinder we use is 90 cm long with a diameter of 13.5 cm.)</li>
	<li>Insert weigh dish with a thin layer of mineral oil underneath the flight cylinder.</li>
	<li>Secure funnel to Ring Stand 2 using a ring clamp and claw clamp. Adjust the height of the funnel so that the bottom of the funnel is flush with the top of the flight cylinder. (Note: the tip diameter of the funnel must be less than the outer diameter of vials placed into the drop tubes so that the vials will not fall through.)</li>
	<li>Insert drop tube into the top of the funnel and secure using a claw clamp. 
	(Note: We use a drop tube that is 25 cm long. Dropping fly-containing vials from this height allows consistent ejection of all flies with uniform force. The inner diameter of the drop tube should be slightly larger than the outer diameter of the vial to allow the vial to drop freely.)</li>
	<li>Cut polyacrylamide sheet(s) to the proper size. (Note: To aid in inserting and removing the sheet, the width should be slightly smaller than the inner circumference of the flight cylinder).</li>
	<li>Apply a thin layer of Tangle-Trap to the sheet. Allow to sit for 1 hr before use. (Note: Leave enough room at the top and bottom of the sheet (approximately&#160;3 cm) uncoated to grasp the sheet for insertion/removal.)</li>
	<li>Insert the polyacrylamide sheet in the flight cylinder.</li>
	<li>Assemble the camera track using pine support brackets. (Note: ensure that the bottom of the track can support the camera without blocking the lens. Refer to Figure 1B.)</li>
	<li>Add stoppers and screw into place. (Note: place the stoppers in locations that will allow the camera to view the entire plastic sheet in panoramic mode.)</li>
</ol>
2. Run Experiment
<ol>
	<li>Collect vials of flies to be tested. For best results, use no more than 20 flies/vial.</li>
	<li>Gently tap flies to bottom of vial, unplug, then insert into drop tube and release vial. 
	(Note: The vial falls down the drop tube until it hits the narrow funnel opening. When the vial hits the funnel, the flies are ejected into the flight cylinder.)</li>
	<li>Lift the drop tube to remove the empty vial. 
	(Note: Multiple vials of flies of the same test group can be assayed on a single polyacrylamide sheet. We find that up to 200 flies (10 vials of 20 flies each) can be tested and imaged readily on a single sheet.</li>
	<li>Remove the plastic sheet and place it on a flat white surface. 
	(Note: white poster board may be used if bench tops are dark colored.)</li>
	<li>Assemble the camera track over the plastic sheet. The camera should be sufficiently high above the sheet to have both the top and bottom of the sheet in the field of view.</li>
	<li>Slide the camera along the track while holding the &#34;capture&#34; button to acquire a panoramic image.</li>
	<li>The number of flies landing in the oil may be counted manually for each trial.</li>
	<li>Repeat steps 2.2-2.7 for all conditions in a given experiment. The flies can be removed from the sheet between each trial. Alternatively, several sheets may be used, with a new sheet for each trial.</li>
</ol>
3. Data Collection
<ol>
	<li>Open image files using ImageJ software.</li>
	<li>Crop images if necessary to include only the landing surface area. (This is the area coated in Tangle-Trap.)</li>
	<li>Convert images to 8-bit grayscale.</li>
	<li>Create a &#34;Threshold&#34; to filter out the white background. 
	(Image &#8594; Adjust &#8594; Threshold).</li>
	<li>Set the parameters to identify each fly using the &#34;analyze particles&#34; menu. 
	(Analyze &#8594; Analyze Particles) Define the parameters used to identify a particle. With our set up, we find that using an area of 5-90 pixels2 and a circularity of 0.4-1.0 will accurately identify all samples.</li>
	<li>Measure the location of each fly using the generated list of coordinates for each particle. The x-coordinate in pixels can be converted to centimeters to calculate the landing height.</li>
	<li>Import table into a spreadsheet (such as Microsoft Excel).</li>
</ol>

<ol>
	<li>Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+behavior.">Genetic dissection of behavior.</a> Sci. Am. 229, 24-37 (1973).</li><li>Ali, Y. O., Escala, W., Ruan, K., Zhai, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assaying+locomotor,+learning,+and+memory+deficits+in+Drosophila+models+of+neurodegeneration.">Assaying locomotor, learning, and memory deficits in Drosophila models of neurodegeneration.</a> J. Vis. Exp. , e2504 (2011).</li><li>de Vries, S. E., Clandinin, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optogenetic+Stimulation+of+Escape+Behavior+in+Drosophila+melanogaster.">Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster.</a> J. Vis. Exp. , e50192 (2013).</li><li>Mundiyanapurath, S., Certel, S., Kravitz, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studying+aggression+in+Drosophila+(fruit+flies).">Studying aggression in Drosophila (fruit flies).</a> J. Vis. Exp. , e155 (2007).</li><li>Nichols, C. D., Becnel, J., Pandey, U. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+to+assay+Drosophila+behavior.">Methods to assay Drosophila behavior.</a> J. Vis. Exp. , e3795 (2012).</li><li>Shiraiwa, T., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proboscis+extension+response+(PER)+assay+in+Drosophila.">Proboscis extension response (PER) assay in Drosophila.</a> J. Vis. Exp. , e193 (2007).</li><li>Lloyd, T. E., Taylor, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flightless+flies:+Drosophila+models+of+neuromuscular+disease.">Flightless flies: Drosophila models of neuromuscular disease.</a> Ann. N.Y. Acad. Sci. 1184, e1-e20 (2010).</li><li>Atkinson, N. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+separation+of+two+behavioral+phenotypes+by+a+mutation+affecting+the+promoters+of+a+Ca-activated+K+channel.">Molecular separation of two behavioral phenotypes by a mutation affecting the promoters of a Ca-activated K channel.</a> J. Neurosci. 20, 2988-2993 (2000).</li><li>Atkinson, N. S., Robertson, G. A., Ganetzky, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+component+of+calcium-activated+potassium+channels+encoded+by+the+Drosophila+slo+locus.">A component of calcium-activated potassium channels encoded by the Drosophila slo locus.</a> Science. 253, 551-555 (1991).</li><li>Elkins, T., Ganetzky, B., Wu, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+mutation+that+eliminates+a+calcium-dependent+potassium+current.">A Drosophila mutation that eliminates a calcium-dependent potassium current.</a> Proc. Natl. Acad. Sci. U.S.A. 83, 8415-8419 (1986).</li><li>Gargano, J. W., Martin, I., Bhandari, P., Grotewiel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+iterative+negative+geotaxis+(RING):+a+new+method+for+assessing+age-related+locomotor+decline+in+Drosophila.">Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila.</a> Exp. Gerontol. 40, 386-395 (2005).</li><li>Fry, S. N., Rohrseitz, N., Straw, A. D., Dickinson, M. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visual+control+of+flight+speed+in+Drosophila+melanogaster.">Visual control of flight speed in Drosophila melanogaster.</a> J. Exp. Biol. 212, 1120-1130 (2009).</li><li>Mronz, M., Lehmann, F. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+free-flight+response+of+Drosophila+to+motion+of+the+visual+environment.">The free-flight response of Drosophila to motion of the visual environment.</a> J. Exp. Biol. 211, 2026-2045 (2008).</li></ol>

The authors have no conflicts of interest to disclose.

Using the methods described here, we have been able to rapidly assess flight performance of a large number of Drosophila mutants, providing greater efficiency than previously possible. For our experiments, we routinely separate males and females and raise them at low density (less than 20 flies/vial) to limit aggression that could damage wings. Another important consideration is to control properly for differences in flight performance owing to differences in genetic background. We also find it helps to allow fl...

Drosophila has long been used to study the genetic basis of behavior1, and researchers have devised a number of ways to analyze various types of behavior2-6. Flies have been particularly useful in providing useful models of neuromuscular disorders7. A common assay used to study locomotor behavior is flight performance. The original flight tester is useful for identifying flight defective mutants and for quantitative assessment of flight ability1, but it has several shortcomings that limit its application for high throughput screens: the use of oil-coated cylinders is messy and cumbersome, certain features such as the length of the cylinder and introduction of flies into the tube with variable force reduce quantitative accuracy; and it is difficult to recover live flies from the tester. To overcome these limitations, we have modified the flight tester to include a number of improvements. We added a &#34;drop tube&#34; to introduce flies to eliminate variability between experiments and users. We use removable acrylic sheets coated with an adhesive that allows for easier cleanup and recovery of individual flies. We have increased the length of the flight tube to improve quantitative accuracy and reliability. Finally, we use a digital camera and imaging software to calculate the landing heights of flies. We believe these improvements will be useful to any laboratory interested in conducting large-scale genetic screens for defects in flight performance.

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			<td>http://rsb.info.nih.gov/ij/</td>
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			<td height="33" style="height:33px;width:216px;">digital camera</td>
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an improved method for accurate and rapid measurement of flight performance in drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

Figure 1A shows a schematic of the updated flight tester assembly. Figure 1B illustrates the track design allow the camera to take a panoramic image without blocking the field of view. Representative results are shown in Figure 2, where the flight performance of slowpoke mutant flies, which have a known flight defect8-10, are compared to wild-type Canton-S flies. Control flies consistently land near the top of the cylinder, with very little s...

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An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

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11.6K Views.  University of Wisconsin-Madison. Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.